Abstract

An epithelial-mesenchymal transition (EMT) is a fundamental example of cell plasticity, involving a reversible switch from epithelial to mesenchymal cell states. An EMT endows stationary epithelial cells with migratory and invasive potential, leads to intravasation into the blood circulation and extravasation to the distant organ. To promote metastatic outgrowth, mesenchymal cells undergo a reverse process of mesenchymal-epithelial transition (MET). Therefore, it is important to understand the underlying mechanisms of epithelial-mesenchymal plasticity to design effective therapeutic approaches that revert EMT and prevent tumor cell invasion and metastasis. The dynamic cell state transitions during EMT imply a role for chromatin rearrangements that are established by epigenetic regulators. However, we still do not fully understand the differences between the epigenetically regulated mechanisms defining the transient cell state transitions of a reversible EMT and the fixed cell status of an irreversible EMT. To delineate these differences, we have generated from murine mammary epithelial cancer cells a novel in vitro irreversible EMT model as compared to a reversible EMT model. Reversible EMT is induced by TGF-β, a potent inducer of EMT. Upon removal of TGF-β, mesenchymal cells undergo a MET and revert to the epithelial state. In contrast, in vitro irreversible EMT cells maintain their mesenchymal state even after removal of the EMT-inducing growth medium. These EMT systems have provided us a unique opportunity to identify the de novo established epigenetic modifiers which maintain the mesenchymal state. Gene expression analysis has revealed a remarkable difference between the reversible and the irreversible EMT systems. Interestingly, irreversible EMT cells exhibit a highly aggressive phenotype in terms of tumor growth rate and metastasis formation as compared to the reversible EMT cells. To identify the epigenetic regulators contributing to the maintenance of the mesenchymal cell state and the aggressive phenotype of irreversible EMT cells, we have used several pharmacological inhibitors targeting various epigenetic modifiers. We have found that histone deacetylase (HDAC) inhibitors partially revert irreversible EMT cells into an epithelial cell state. Due to the merely partial contribution of HDACs to an irreversible EMT, we have further explored additional contributors to the maintenance of the mesenchymal cell state. HDACs are involved in several corepressor complexes to exert their specific functions. The Mbd3/NuRD complex is one of the corepressor complexes containing HDAC1/2. It plays an important role in the generation of induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs), indicating a key role in cellular plasticity. Notably, Mbd3 is the only methyl binding domain protein which is not able to bind to the methylated cytosines due to an amino acid substitution in the methyl binding domain. Instead, it is thought that it recognizes the DNA demethylation intermediate 5-hydroxymethyl cytosine which is generated by Tet hydroxylases. Using loss of function experiments, we demonstrate that the Mbd3/NuRD complex, involving histone deacetylases (HDACs) and Tet2 hydroxylase, acts as an epigenetic block in epithelial-mesenchymal plasticity. Interestingly, these epigenetic factors keep the mesenchymal cells in a stable state and promote the aggressive cancer cell phenotype by regulating a wide-range of gene networks. The pharmacological inhibition of HDACs and ablation of Mbd3 and/or Tet2 leads to a MET as well as to diminished tumor growth and metastasis formation. These results provide important insights into the epigenetic regulation of epithelial-mesenchymal plasticity and identify novel therapeutic targets to interfere with primary tumor growth and metastasis formation. In particular, the development of specific inhibitors of Tet hydroxylases and their combinatorial use with HDAC inhibitors may be an effective therapeutic approach to prevent tumor progression and metastasis.